This application is a 371 of PCT/GB/94/02695 filed Nov. 14, 1994.
The present invention relates to preparations of substances in hydrophobic solvents in which they would not normally be soluble and to processes for obtaining these preparations. In particular, the invention relates to preparations of hydrophilic species in hydrophobic solvents such as oils.
The invention in particular applies to hydrophilic macromolecules which would not normally be soluble in oils or other hydrophobic solvents.
For many applications, e.g in the pharmaceutical sciences, in food technology or the cosmetics industry, work with proteins and similar macromolecules presents problems because their hydrophilicity and high degree of polarity limit the extent to which they can interact with or incorporate into lipid phases. Many natural systems employ lipidic barriers (eg skin, cell membranes) to prevent access of hydrophilic molecules to internal compartments; the ability to disperse proteins in lipidic vehicles would open up a new route to introduction of these macromolecules into biological systems, whereby the lipid medium containing the protein can integrate with the hydrophobic constituents of barriers, instead of being excluded by them.
Another area where dissolution of proteins into oils may confer advantage is for the use of enzymes in organic phases. Enzymic syntheses are becoming increasingly important compared to chemical processes because of their much lower energy needs, greater substrate and product specificities, high yields, and the fact that many reactions are catalysed which are impossible by chemical means. Recent findings that enzymes can remain active in organic environments have opened up many additional possibilities. Thus, reactions involving lipophilic substrates and products may be catalysed effectively, and enzyme stability is often much greater than in aqueous environments, allowing them to be used in much more extreme conditions such as at high temperature. A very important aspect is that reactions involving hydrolytic enzymes such as lipases and peptidases can preferentially go in the reverse direction in low water environments, thus enabling the synthesis of a wide range of industrially important compounds. Another application is where a complex chain of reactions is involved in which the multiple catalytic units need to be maintained in close proximity to each other. Such might be the case in light-initiated redox reactions. An additional possibility is the controlled production of nanoparticulates in oil phase, using enzymes to induce (mineralisation by action on organometallic substrates. The preparation of a stable dispersion of preformed nanoparticulates in oil phase may also be advantageous for the performance of certain surface-catalysed reactions.
Dispersion of hydrophilic substances in oil phase rather than aqueous media confers other benefits in terms of increasing their stability with respect to temperature-mediated denaturation, hydrolysis, light sensitivity etc. Oils can be chosen which remain fluid over a wider temperature range than aqueous solutions, or that have a higher viscosity, resulting in greater protection against physical damage. In mixed-phase systems, sequestration of proteins in oil can limit mutually harmful interactionsxe2x80x94eg oxidationxe2x80x94with water-soluble compounds.
There are examples of formulations containing both macromolecules and oil and one such example is disclosed in EP-A-0366277. The formulation disclosed in this document is an emulsion having both a hydrophobic and a hydrophilic phase, wherein the hydrophobic phase contains chylomicra or chylomicron-forming lipids. However, the macromolecule is dissolved in the hydrophilic phase not in the hydrophobic phase.
EP-A-0521994 also relates to a composition suitable for the oral delivery of macromolecules which comprises a biologically active material in association with lecithin or a compound capable of acting as a precursor for lecithin in vivo. All of the compositions exemplified are formulations which comprise a hydrophilic and a lipophilic phase. Once again, in this prior art document, the macromolecule is dissolved in the hydrophilic phase rather than in the lipophilic phase.
Although the formulations mentioned above do contain both macromolecules and oils, it is significant that in all cases the macromolecule is dissolved in the hydrophilic rather than in the lipophilic phase. Attempts to form true solutions of macromolecules in oils have met with limited success.
The present invention relates to the surprising discovery that if a hydrophilic species is mixed with an amphiphile under certain conditions, the resultant composition will be readily soluble in lipophilic solvents as oils.
In a first aspect of the present invention there is provided a process for the preparation of a single phase hydrophobic preparation comprising a hydrophilic species, in a hydrophobic solvent, the process comprising:
(i) associating the hydrophilic species with an amphiphile in a liquid medium such that, in the liquid medium, there is no chemical interaction between the amphiphile and the hydrophilic species;
(ii) removing the liquid medium to leave an array of amphiphile molecules with their hydrophilic head groups orientated towards the hydrophilic species; and
(iii) providing a hydrophobic solvent around the hydrophilic species/amphiphile array.
In the context of the present invention, the term xe2x80x9cchemical interactionxe2x80x9d relates to an interaction such as a covalent or ionic bond or a hydrogen bond. It is not intended to include van der Waals forces or other interactions of that order of magnitude.
It has been found that the order in which the components of the preparation are mixed is particularly important. In one attempt to prepare a molecular dispersion, we mixed a macromolecular compound (an example of a hydrophilic species) with the hydrophobic solvent and then added amphiphile whilst in an alternative procedure, a macromolecular compound was added to a mixture of the hydrophobic solvent and the amphiphile. However, both of these approaches result in the production of a grainy dispersion of the macromolecular compound in the solvent rather than in a true molecular dispersion. It was found that only by adding the macromolecular compound to the amphiphile in such a way that an array is produced in which the hydrophilic head groups of the amphiphile are orientated towards the macromolecule and then dissolving this array in the hydrophobic solvent, could a single phase preparation be produced.
As mentioned above, the hydrophilic species and the amphiphile are associated in a liquid medium and in many cases the array is formed in the liquid medium before it is removed. This occurs when the amphiphile and liquid medium are such that the array is formed in the liquid medium even in the absence of a hydrophilic species.
In the present invention the term xe2x80x9chydrophilic speciesxe2x80x9d relates to any species which is generally soluble in aqueous solvents but insoluble in hydrophobic solvents. The range of hydrophilic species of use in the present invention is diverse but hydrophilic macromolecules represent an example of a species which may be used.
A wide variety of macromolecules is suitable for use in the present invention. In general, the macromolecular compound will b e hydrophilic or will at least have hydrophilic regions since there is usually little difficulty in solubilising a hydrophobic macromolecule in oily solutions. Examples of suitable macromolecules include proteins and glycoproteins, oligo and polynucleic acids, for example DNA and RNA, polysaccharides and supramolecular assemblies of any of these including, in some cases, whole cells or organelles. It may also be convenient to co-solubilise a small molecule such as a vitamin in association with a macromolecule, particularly a polysaccharide such as a cyclodextrin. Small molecules such as vitamin B12 may also be chemically conjugated with macromolecules and may thus be included in the compositions.
Examples of particular proteins which may be successfully solubilised by the method of the present invention include insulin, calcitonin, haemoglobin, cytochrome C, horseradish peroxidase, aprotinin, mushroom tyrosinase, erythropoietin, somatotropin, growth hormone, growth hormone releasing factor, galanin, urokinase, Factor IX, tissue plasminogen activator, superoxide dismutase, catalase, peroxidase, ferritin, interferon, Factor VIII and fragments thereof (all of the above proteins can be from any suitable source). Other macromolecules may be used are FITC-labelled dextran and RNA extract from Torulla yeast.
It seems that there is no upper limit of molecular weight for the macromolecular compound since dextran having a molecular weight of about 1,000,000 can easily be solubilised by the process of the present invention.
In addition to macromolecules, the process of the present invention is of use in solubilising smaller organic molecules. Examples of small organic molecules include glucose, carboxyfluorescin and many pharmaceutical agents, for example anti-cancer agents, but, of course, the process could equally be applied to other small organic molecules, for example vitamins or pharmaceutically or biologically active agents. In addition, compounds such as calcium chloride and sodium phosphate can also be solubilised using this process. Indeed, the present invention would be particularly advantageous for pharmaceutically and biologically active agents since the use of non aqueous solutions may enable the route by which the molecule enters the body to be varied, for example to increase bioavailability.
Another type of species which may be included in the hydrophobic compositions of the invention is an inorganic material such as a small inorganic molecule or a colloidal substance, for example a colloidal metal. The process of the present invention enables some of the properties of a colloidal metal such as colloidal gold, palladium, platinum or rhodium, to be retained even in hydrophobic solvents in which the particles would, under normal circumstances, aggregate. This could be particularly useful for catalysis of reactions carried out in organic solvents.
A process somewhat similar to that of the present invention is disclosed by Okahata et al (J. Chem. Soc. Chem. Commun., 1988, 1392-1394). However, it seems that the array of protein surrounded by amphiphile molecules produced by the authors of that document differed considerably from that produced by the method of the present invention. In particular, the authors stated that the amphiphile molecules reacted with the protein in the liquid medium by hydrogen bonding or via an electrostatic interaction to form a solid precipitate. In contrast, it seems that in the present invention the hydrophilic species does not interact chemically with the amphiphile molecules in the liquid medium.
There are numerous amphiphiles which may be used in the present invention and zwitterionic amphiphiles such as phospholipids are among those which have been found to be especially suitable. Phospholipids having a phosphatidyl choline head group have been used with particular success and examples of such phospholipids include phosphatidyl choline (PC) itself, lyso-phosphatidyl choline (lyso-PC), sphingomyelin, derivatives of any of these, for example hexadecylphosphocholine or amphiphilic polymers containing phosphoryl choline. In the present application, the terms phosphatidyl choline (PC) and lecithin are used interchangeably. Suitable natural lecithins may be derived from any convenient source, for example egg and, in particular, soya. In most cases, it is preferable to select an amphiphile which is chemically similar to the chosen hydrophobic solvent and this is discussed in greater detail below.
The fact that the present inventors have found zwitterionic amphiphiles such as phospholipids to be particularly suitable for use in the process is a further indication of the significant differences between the present invention and the method of Okahata et al. Significantly, the authors of that prior art document concluded that anionic and zwitterionic lipids were completely unsuitable for use in their method and stated that they obtained zero yield of their complex using these lipids.
The hydrophobic solvent of choice will depend on the purpose for which the composition is intended, on the type of species to be solubilised and on the amphiphile. Suitable solvents include long chain fatty acids with unsaturated fatty acids such as oleic and linoleic acids being preferred, alcohols, particularly medium chain alcohols such as octanol and branched long chain alcohols such as phytol, monoglycerides such as glycerol monooleate (GMO), diglycerides and triglycerides, particularly medium chain triglycerides and mixtures thereof.
Optimum results are generally obtained when the hydrophobic solvent and the amphiphile are appropriately matched. For example, with a solvent such as oleic acid, lyso-PC is a more suitable choice of amphiphile than PC, whereas the converse is true when the hydrophobic solvent is a triglyceride.
In addition, in some cases it has been found to be advantageous to add a quantity of the amphiphile to the hydrophobic solvent before it is brought into contact with the hydrophilic species/amphiphile array. This ensures that the amphiphile molecules are not stripped away from their positions around the hydrophilic species because of the high affinity of the amphiphile for the hydrophobic solvent.
It is very much preferred that the preparations of the invention are optically clear and this can be monitored by measuring turbidity at visible wave lengths and, in some cases, by checking for sedimentation over a period of time.
A hydrophile/amphiphile array in which the hydrophilic head groups of an amphiphile are orientated towards a hydrophilic species has been produced before but it has never been suggested that this type of composition may be soluble in lipophilic solvents.
Kirby et al, in Bio/Technology, November 1984, 979-984 and in Liposome Technology, Volume I, pages 19-27, Gregoriadis, Ed., CRC Press, Inc., Boca Raton, Fla., USA describe a method for the preparation of liposomes in which a phospholipid is suspended in distilled water to form small unilamellar vesicles or multilamellar vesicles, mixed with the material to be entrapped and freeze dried. The mixture is then rehydrated to give liposomes.
At the time of publication of this prior art there was extensive worldwide interest in the preparation of liposomes but the idea of producing a single phase hydrophobic preparation of a macromolecule seems either never to have been thought of or to have been dismissed as impossible or of little value. Certainly, there is no suggestion in any of the prior art that the intermediate arrays could be put to any other use than the preparation of liposomes. Even if a single phase hydrophobic preparation had been a desirable objective, the idea of adding a hydrophobic rather than a hydrophilic solvent would have been unlikely to have been taken seriously because there was a strong prejudice in the art against hydrophobic preparations of hydrophilic molecules.
The orientation of amphiphile molecules into an array with their hydrophilic head groups facing the moieties of a hydrophilic species can be achieved in several ways and examples of particularly suitable methods are discussed in more detail below.
In a first method, which has a similar starting point to the method described by Kirby et al, supra, a hydrophilic species is mixed with a dispersion of an amphiphile in a hydrophilic solvent, such that the amphiphile molecules form an assembly in which the hydrophilic head groups face outwards towards the hydrophilic phase which contains the hydrophilic species. The hydrophilic solvent is then removed to leave a dry composition in which the hydrophilic head groups of the amphiphile molecules are orientated towards the hydrophilic species.
In the method described by Okahata et al, a solution of a protein was also mixed with a dispersion of an amphiphile in water. However, significantly, the authors of that paper believed that it was necessary to obtain a precipitate which would then be soluble in hydrophobic solvents. Since many of the preferred amphiphiles of the present invention do not form such a precipitate, Okahata et al concluded that they would be of no use. In the process of the present invention, no precipitate is required and, indeed, it is generally thought to be undesirable to allow the formation of a precipitate since this results in a reduced yield of the required product.
In this first method, it is preferred that the hydrophilic solvent is water although other polar solvents may be used.
The form taken by the amphiphile assembly may be micelles, unilamellar vesicles, preferably small unilamellar vesicles which are generally understood to have a diameter of about 25 nm, multilamellar vesicles or tubular structures, for example cochleate cylinders, hexagonal phase, cubic phase or myelin type structures. The form adopted will depend upon the amphiphile which is used and, for example, amphiphiles such as phosphatidyl choline (PC) tend to form small unilamellar vesicles whereas lyso-phosphatidyl choline forms micelles. However, in all of these structures, the hydrophobic tails of the amphiphile molecules face inwards towards the centre of the structure while the hydrophilic head groups face outwards towards the solvent in which the hydrophilic species is dispersed.
The weight ratio of amphiphile:hydrophilic species will generally be in the region of from 1:1 to 100:1, preferably from 2:1 to 20:1 and most preferably about 8:1 for PC and 4:1 for lyso-PC.
These ratios are preferred ratios only and, in particular, it should be pointed out that the upper limit is set by economic considerations which mean that it is preferable to use the minimum possible amount of amphiphile. The lower limit is somewhat more critical and it is likely that ratios of 2:1 or below would only be used in cases where the hydrophilic species has a significant hydrophobic portion or is exceptionally large.
Good performance is obtained when the solvent is removed quickly and a convenient method for the removal of the solvent is lyophilisation, although other methods can be used.
In some cases, it may be helpful to include salts in the hydrophilic solution, particularly if the hydrophilic species is a macromolecular compound such as a large protein. However, because the presence of larger amounts of inorganic salts tends to give rise to the formation of crystals and, hence, to a cloudy solution, it is preferred that organic salts are used rather than inorganic salts such as sodium chloride. Ammonium acetate is especially suitable for this purpose since it has the additional advantage that it is easily removed by freeze drying.
A second method for the preparation of a composition containing an array of amphiphiles with their head groups pointing towards the moieties of the hydrophilic species is to co-solubilise the hydrophilic species and the amphiphile in a common solvent followed by removal of the solvent.
This second method of forming the array is novel and itself forms a part of the invention.
Therefore, in a second aspect of the invention there is provided a process for forming a hydrophile/amphiphile array wherein the hydrophilic head groups of the amphiphile molecules are orientated towards the hydrophilic species, the process comprising co-solubilising a hydrophilic species and an amphiphile in a common solvent and subsequently removing the common solvent.
When this method is used, it is preferred that the weight ratio of amphiphile:hydrophilic species is from about 1:1 to 50:1, preferably from 2:1 to 10:1 and most preferably about 4:1.
The common solvent must, of course, dissolve both the amphiphile and the hydrophilic species and will, for preference, be a polar organic solvent such as dimethylformamide, dimethylsulphoxide or, most suitably, glacial acetic acid.
In this method, in contrast to the first method, it is unlikely that an array will be formed before the removal of the common solvent.
It seems probable that, on removal of the solvent, the amphiphile molecules tend to order themselves in sheets with their head groups towards the hydrophilic species and their lipophilic tail groups facing away from the hydrophilic species. However, the effectiveness of the present invention does not depend on the accuracy or otherwise of this observation.
It has been observed that good results are obtained when the solvent is removed slowly, for example by drying under a stream of nitrogen, probably because this allows more time for the amphiphile molecules to reorder themselves.
A third method for forming the hydrophile/amphiphile array comprises emulsifying a solution of the amphiphile in a hydrophobic solvent with a solution of the hydrophilic species in a hydrophilic solvent to give an emulsion, and removing the solvents.
The emulsion may be either a water-in-oil or an oil-in-water type, but if a small hydrophilic species is used rather than a macromolecule, then a water-in-oil emulsion may be more suitable.
Any hydrophobic solvent for the amphiphile may be used, but for the water-in-oil emulsions preferred for use with small hydrophilic species, a low boiling point solvent such as diethyl ether is preferred since it has been found that the best results are obtained when the hydrophobic solvent is removed slowly by gentle methods such as evaporation and, clearly, this is most effective using a low boiling point solvent. Low boiling point solvents are also preferred for water-in-oil emulsions although, for these, lyophilisation is a more suitable method of solvent removal. The hydrophilic solvent will preferably be aqueous.
The weight ratio of amphiphile:hydrophilic species may be from about 1:1 to 50:1, preferably from 2:1 to 10:1 and most preferably about 4:1.
The ratio of hydrophilic solution to hydrophobic solution is not critical, but if small hydrophilic species are used, it is preferably such as to ensure the formation of a water-in-oil emulsion rather than an oil-in-water emulsion.
When a water-in-oil emulsion is formed, the third method is suitable for use with any type of hydrophilic species but the first and second methods have been found to be less suited to use with small molecules than the third method.
An alternative method of forming the array, which may be particularly suited to use with small hydrophilic species, is to entrap the hydrophilic species in closed lipid vesicles such as small unilamellar vesicles (SUVs) dispersed in a hydrophilic solvent and then to remove the solvent.
The product of the process of the invention is new since it makes possible the production of single phase hydrophobic preparations comprising a hydrophilic species which would not normally be soluble in a hydrophobic solvent. Therefore, in a third aspect of the invention there is provided a single phase hydrophobic preparation comprising a hydrophilic species in a hydrophobic solvent obtainable by the process of the invention.
Additionally, the present invention also provides a single phase hydrophobic preparation comprising a hydrophilic species and an amphiphile in a hydrophobic solvent, characterised in that the moieties of the hydrophilic species are surrounded by amphiphile molecules with the hydrophilic head groups of the amphiphile molecules orientated towards the hydrophilic species.
Preferred hydrophilic species, amphiphiles and hydrophobic solvents are as specified for the process just described.
It may also be desirable to include other constituents in the single phase hydrophobic preparation in addition to the hydrophilic species. This is often particularly appropriate when the hydrophilic species is a macromolecule and, in that case, the preparation may include, for example, bile salts, vitamins or other small molecules which bind to or are otherwise associated with the macromolecules.
Although some macromolecule/amphiphile arrays were disclosed by Kirby et al, supra, the arrays disclosed were all intermediates in the formation of liposomes and, as discussed above, there has been no previous interest in non-liposomal or hydrophobic compositions comprising this type of entity. Therefore, the arrays of the present invention in which the amphiphile is one which does not form small unilamellar vesicles and would therefore not be expected to form liposomes are new.
In a further aspect of the invention there is provided an array of amphiphile molecules and hydrophilic species characterised in that the hydrophilic head groups of the amphiphile molecules are orientated towards the hydrophilic species and wherein there is no chemical interaction between the amphiphile and the hydrophilic species, provided that the amphiphile is one which is not capable of forming liposomes when water is added to the array.
One example of an amphiphile which is not capable of forming liposomes is lyso-lecithin. In most aqueous environments, this amphiphile forms micelles rather than small unilamellar vesicles and it is therefore unsuitable for use in the preparation of liposomes. It is however extremely useful in the process of the present invention, particularly when used in conjunction with a compatible hydrophobic solvent such as oleic acid.
One advantage of the preparations of the present invention is that they are essentially anhydrous and therefore stable to hydrolysis. They are also stable to freeze-thawing and have greater stability at high temperatures, probably because water must be present in order for the protein to unfold and become denatured. This means that they may be expected to have a much longer shelf life than aqueous preparations of the hydrophilic species.
The solutions of the present invention are extremely versatile and have many applications. They may either be used alone or they may be combined with an aqueous phase to form an emulsion or similar two phase composition which forms yet a further aspect of the invention.
In this aspect of the invention there is provided a two phase composition comprising a hydrophilic phase and a hydrophobic phase, the hydrophobic phase comprising a preparation of a hydrophilic species in a lipophilic solvent obtainable by a process as described herein.
Generally, in this type of composition, the hydrophobic phase will be dispersed in the hydrophilic phase.
It is surprising that a stable two phase composition of this type can be formed since it might have been expected that the hydrophilic species would not remain in the hydrophobic phase but would, instead, pass to the hydrophilic phase. However, it has been demonstrated that in many cases this does not occur and that the hydrophilic species does, indeed, remain in association with the dispersed hydrophobic phase and is not present in free solution. It is possible that this is a result of some residual water or other hydrophilic remaining bound to the hydrophilic head group of the amphiphilic molecule. One advantage of this is that osmotic leakage of the hydrophilic species is not a problem in the compositions of the invention as is the case with some known systems, particularly liposomal systems.
The two phase compositions may be emulsions which may either be transient or stable, depending on the purpose for which they are required.
The average size of the emulsion particles will depend on the exact nature of both the hydrophobic and the aqueous phases. However, it may be in the region of 2 xcexcm
Dispersion of the hydrophobic preparation in the aqueous phase can be achieved by mixing, for example either by vigourous vortexing for a short time for example about 10 to 60 seconds, usually about 15 seconds, or by gentle mixing for several hours, for example using an orbital shaker.
Emulsions containing the hydrophobic preparations of the invention can also be used in the preparation of microcapsules. If the emulsion is formed from a gelatin-containing aqueous phase, the gelatin can be precipitated from the solution by coacervation by known methods and will form a film around the droplets of the hydrophile-containing hydrophobic phase. On removal of the hydrophilic phase, microcapsules will remain. This technology is known in the art, but has proved particularly useful in combination with the preparations of the present invention.
One way in which the compositions of the present invention may be used is for the oral delivery to mammals, including man, of substances which would not, under normal circumstances, be soluble in lipophilic solvents. This may be of use for the delivery of dietary supplements such as vitamins or for the delivery of biologically active substances, particularly proteins or glycoproteins, including insulin and growth hormones.
In a further application, it is possible to encapsulate or microencapsulate, for example by the method described above, nutrients such as vitamins which can then be used, not only as human food supplements but also in agriculture and aquaculture, one example of the latter being in the production of a food stuff for the culture of larval shrimps.
In addition, the compositions find application in the preparation of pharmaceutical or other formulations for parenteral administration, as well as formulations for topical or ophthalmic use. For this application, it is often preferable to use an emulsion of the oil solution and an aqueous phase as described above.
Many therapeutic and prophylactic treatments are intended for sustained or delayed release or involve a two component system, for example including a component for immediate release together with a component for delayed or sustained release. Because of their high stability, the preparations of the invention are particularly useful for the formulation of a macromolecule intended for sustained or delayed release.
The longer shelf life of the compositions of the present invention is a particular advantage in the pharmaceutical area.
The hydrophile-in-oil preparations may find application in the pharmaceutical or similar industries for flavour masking. This is a particular problem in the pharmaceutical industry since many drugs have unpleasant flavours and are thus unpopular with patients, especially children.
A further use is in the cosmetics industry where, again, hydrophobic preparations of hydrophilic compounds can very easily be incorporated into a cosmetic formulation. Examples of macromolecules which may be used in this way include those with moisturising or enzymatic action of some sort. The invention can also be used for the incorporation of proteins such as collagen into dermatological creams and lotions.
Finally, the invention has numerous uses in the field of chemical and biological synthesis, for example, non-aqueous enzymatic synthesis.